generation and exploitation of marine natural product
TRANSCRIPT
Generation and Exploitation of Marine Natural Product Extract Collections as
Source of New Bioactive Molecules
Fundación MEDINANovember 2018
Discovering the Future
Fernando Reyes, PhDHead of Chemistry
• Introduction of Fundación MEDINA and ourcollection
• Creation of Natural Product ExtractsCollections
• Exploitation of collections in search of newbioactive molecules
Outline
Independent, Non-profit Research Organization established as a Private-public partnership between:
• Government of Andalucía (Spain)
• University of Granada (Spain)
• Merck Sharp and Dohme de España S.A.
Our Mission
Discovery of new bioactive compounds and innovative therapies
for unmet medical and industrial needs.
ABOUT US
General Hospital
Faculty of Medicine
Bio Incubator
UGR-I.Biomedicine
CSIC- I. Lopez Neyra
MEDINA
Genyo
HEALTH SCIENCES TECHNOLOGY PARK, GRANADA, SPAIN
Faculty of Health Sciences
MEDINA:Microbial Natural Products Drug Discovery
• Multidisciplinary team of
scientists (28 permanent staff ,
up to 40 employees)
• 2300 m2 fully equipped
facilities
• One of the largest microbial
collections and libraries for
natural products drug discovery
• High capacity center for
high throughput screening
• Drug discovery research
programs in infectious
diseases, cancer and
neurodegeneration
●Microbial collections (190.000 strains)
●Microbial Genome Banks
●Natural Products Libraries (200.000 extracts)
●New Compound Discovery
●Patents
●Collaborations
●Publications
●High Throughput Screening
●Natural Products Chemistry
●Compound Structural Elucidation
●Bioanalytics
●Preclinical Safety
●Genome mining
MEDINA at a glance
RESEARCH SERVICES
Academia & Industry
Leveraging:
• 60 years expertise in Drug Discovery
• 5 Drugs in the market
• Collaborations with
industry & academia
Contribution of CIBE-MRL Spain to Merck Natural Product Drug Discovery
Thienamycin
(Imipenem, Merck)
Lovastatin
(Mevacor, Merck)
Caspofungin
(Cancidas, Merck)
Leads and clinical candidatesDrugs in the market
Immunomycin*immunosuppressor
O
O
OH
OH
NH
HN
DMAQ-B1*Insulin mimetic
O
O
NH
O
OH
OH
HO2C
Enfumafungin*, antifungal
Platensimycin *, antibacterial
Fatty acid synthesis inhibitor
ON O
O
O OHOH
HO COOCH3HO
N OO
O
COOCH3
OH
HO
O
Kibdelomycin*, antibacterial
Topo II isomerase inhibitor
Parnafungins*mRNA polyadenylation inhibitors
O
O
H
H
O
OH
HO
HOHO
AcO
OH
COOH
MICROBIAL NATURAL PRODUCTS
Maximizing chemical diversity by maximizing biodiversity
Geographical, Ecological & Taxonomical Diversity
* Recent donation of new 74,000 strains from former Cubist-Merck & Co. Inc Collection
Microbial Strain Collections
190,000 strains*Fungi, Actinomycetes & unicellular Bacteria
Natural Products Libraries
>200,000 extractsfrom a large diversity of well-identified microbial strains
Proven genetic and chemical diversity
9
Sources:TerrestrialMarine
LARGEST MICROBIAL COLLECTION WORLDWIDE WITH 190.000 STRAINS
GeographyMarine & Terrestrial
EcologyPlants, roots, sediments,
rocks, water…
TaxonomyFungi, Actinomycetes &
unicellular bacteria
Fungal plant endophytes: 32%Soil & plant rhizosphere actinomycetes: 86%Marine actinomycetes: < 10%
• Introduction of Fundación MEDINA and ourcollection
• Creation of Natural Product ExtractsCollections
• Exploitation of collections in search of newbioactive molecules
Outline
Extracts, fractions & compound Libraries
Smart High Throughput Screening
Hit Confirmation & LC/MS dereplication
Hit Selection
Bioassay-guided Isolation
Structural Elucidation
HPLC-MS & NMR
Natural Products Discovery
Novel Compound
• Known Composition
• Known concentration
[10 mM]
• Vehicle DMSO
• Three freeze-thaw
cycles maximum
• Recurrent
Degradation
analyses required
LC-HRMS (QC)
Purified Natural
Products Libraries
Synthetics/Combin
atorial Libraries
Extracts/Fractions
NP Libraries
Types of HTS Libraries
• Known/unknown
Composition
• Know/unknown
concentration [1 - 5
mg/mL]
• vehicle DMSO
• Three freeze-thaw
cycles maximum
• Degradation
analyses required
LC-HRMS
• Unknown
Composition/
• Potentially new
chemistry
• Unknown
concentration [WBE]
• vehicle DMSO/AQ
• Dereplication required
LC-MS/NMR
• Hit follow-up required
for identification
• Cover a wide and representative range of biological species
• Created using robust and reproducible protocols
• Must represent the most significant biodiversity (ideally all)included in the microbial strains or macroorganism samplecollection
• Ideally a back-up sample or a protocol to generate it must beavailable for scaling-up purposes
• Extracts must be stored in appropriate conditions and in a formatsuitable for the rapid generation of multiple aliquots
• Associated information managed through a LaboratoryInformation Management System (LIMS)
• Samples included in the collection must have been legally collectedin compliance with Nagoya protocol and other national andinternational legislation
Collections of extracts-General Rules
14
Preparation of extracts
EXPEDITIONS
EXTRACTION
Water/MeOH:DCM
COLLECTION
Harvesting and
Logistics
CLASIFICATION
DATA MANAGEMENT
Taxonomy & Labelling
GPS, Images
Storage, Safety
Expeditions, Samples and Extract Collections
200.000 extracts!!!
MARINE BIODISCOVERY IN IRELAND
Academics
Private
Academics
Privates
Marine
Organisms
Vouchers
Biomass
Fractions
Chem and Biol
Profilings
Collection/Sampling
SOP MB-100/101
Sample preparation
SOP MB-102
Chemical Screening
SOP MB-103
Biological Screening
SOP MB-104
Biomass
Voucher
Picture
Fractions
FUNCTIONING OF THE NMBLI
WEBDATABASE: RESTRICTED ACCESS
End 2018: 141 Irish Invertebrates. Voucher and Biomass
Help to organise and structure. 423 Fractions to be tested. Tracking
CMS: OPEN ACCESS www.imbd.ie
Location, species (link WORMS), natural products (link METABOLIGHTS)
Maximizing probabilities of success in NPs drug discovery from microorganisms
HTS Targets
Production conditions
& Gene Expression
Strains & Whole
Genome
Microbial ecology
Metagenomics
Metabolomics
System biology
Synthetic biology
Microbiological Approaches to Maximize Chemical Diversity
20
• Genes encoding biosynthetic
enzymes in bacteria and fungi
outnumber known secondary
metabolites
• We are missing large part of NP
biosynthetic capacity
• Only a subset of pathways is
expressed in laboratory conditions
• Access to this reservoir of
molecules needs activation of
cryptic genes
• Culture conditions (OSMAC)
• Stress factors
• Addition chemical inducers
• Interspecies crosstalk: Co-culturing
• Epigenetic modifiers
“The research leading to these results has received funding from the European Union's Seventh Framework Programme(FP7/2007-2013 under grant agreement n° 312184)”
Geographical Origin of MEDINA´s Marine Strains
Over 5,500
marine strains
Taxonomic Diversity of the Marine Collection
High taxonomic diversity
within the collection, which
includes obligate marine
bacteria such as
Salinispora spp.
Diversity tree: partial 16S rRNA sequences of 1,600 strains
over 5,500 marine bacteria,
mainly actinobacteria (~75%),
non-actinobacteria taxa (~25%).
PharmaSea: Selection of Strains
Geographic Origin
Taxonomy
Microbial Source
Strains were selected in order to maximize the diversity in geographic origin,
taxonomy and isolation source
Fermentation of each strain in 4 different media
Components (in g/L) DEF15S-M APM9-M R358 FPY12-M
Amicase 5
Bacto Peptone 2 5
Bacto Yeast Extract 4
Fructose 20
Glucose 50 10
Maltose 10
Saccharose 5
Soluble Starch (potato) 20 12 10
Soybean Flour 30
CoCl2.6H2O 2 mg
FeSO4.7H2O5 mL
(stock 8 g/L)
KBr5 mL
(stock 8 g/L)
KH2PO4 1
MgCl2.6H2O 1
Na2SO4 2
NaCl 1
NH4Cl 2
CaCO3 2 7
Sigma Sea Salts 40 40 40 40Trace Elements 1 mL 1 mL
pH7.0 (before adding
CaCO3)
7.0 (before adding
CaCO3)7 7
PharmaSea Extraction Procedure Details
50 mLfermentation
Transfer supernatant to
new tube
add resinShake
centrifuge
50mL WB +50 mL AcetoneShake, take 40 mL
evaporate acetoneunder N2
Discard supernatant add water
Shakecentrifuge
add acetone add DMSO
Shakecentrifuge
Discard supernatant
evaporate acetoneunder N2 to 20%
DMSO/water
HPLCanalysis
Metabolomics extract selection
Transfer 4 x 500 uL into HTS plates
Blank MediaSPE-extracts
SPE-extracts
+
CLASSIFY WB-SPEs and blanks
evaporate acetoneto 20% DMSO/water
add water
Transfer 200uLto HTS plate
2X WBE Final concentration
Pharma Sea Collection generated at MEDINA (6K)
A: SPEEXTRACTION
B: POLARITYCHROMATOGRAPHY
Module of
CRUDESModule of
FRACTIONS
Module Type of Sample # Samples
PCA-000001 to PCA-000022 Crudes + blanks 1696
PFA-000001 to PFA-000054 Fractions + blanks 4320
Storage - Labeling
LIMS Tracking of Samples: NAPIS
LIMS Tracking of Samples: Nautilus
Strains
Small scale Fermentations
Fractions
Extracts
Sub-Fractions
Large scale Fermentations
Assay Aliquots
• Introduction of Fundación MEDINA and ourcollection
• Creation of Natural Product ExtractsCollections
• Exploitation of collections in search of newbioactive molecules
Outline
32
Antitumor Screening -Cytotoxicity
ET-743, Trabectedin, Yondelis
Ecteinascidia turbinata
Approved for the treatment of Soft
Tissue Sarcoma and Ovarian Cancer
PM0184
Lithoplocamia lithistoides
Undergoing Phase II clinical trials against
colorectal cancer as single agent
Pharma Sea Collection generated at MEDINA (6K)
A: SPEEXTRACTION
B: POLARITYCHROMATOGRAPHY
Module of
CRUDESModule of
FRACTIONS
Module Type of Sample # Samples
PCA-000001 to PCA-000022 Crudes + blanks 1696
PFA-000001 to PFA-000054 Fractions + blanks 4320
Antibacterial
MEDINA - PharmaSea Antimicrobial screening
Staphylococcus aureus MRSAPseudomonas aeruginosaEscherichia coli β-lactam synergy vs. multi-resistant Pseudomonas aeruginosa
Candida albicansAspergillus fumigatus
Antifungal
Mycobacterium tuberculosis H37Ra Mycobacterium bovis BCG
Anti-TB
This project has received funding from the Novo Nordisk Foundation’s Challenge programme.
3
High Throughput Antibacterial Screening Overview
Microbial
Extracts
Pathogen
Inoculum
Positive and
negative controls
Total Growth
Medium Control
ULTRA TECAN
t0 Absorbance (Growth
density OD= 612nm)
Incubation O/N
at 37oC
ULTRA TECAN
tf Absorbance
(Growth density
OD= 612nm)Data Analysis
HIT
IDENTIFICATION
Zebrafish-based identification of neuroactive
and antiepileptic hits (KU Leuven)
OR 0
50
100
150PTZ
VHC
PS-243 - 100µg/ml
PS-243 - 50µg/ml
PS-243 - 25µg/ml
0-5 5-10 10-15 15-20 20-25 25-30
******
*** ******
** *** *** ***
*
***** ***
time (min)
%P
TZ-i
nd
uced
acti
vit
y
• Photomotor response assay: neuroactive hits
• Epilepsy seizure model: anticonvulsant hits
• Maximum Tolerated Concentration (MTC) analysis: general toxicity analysed for hits
3777 screened samples727 neuroactive hits43 anticonvulsant hits5 out of 11 hits validated
To be continued
Screening Campaigns Results
Count of Hits Qualifier Extracts
per Assay A Q I Tested
Absorbance_MB2884 (E.coli) 68 137 5761 5966
Absorbance_MB5393 (MRSA) 114 10 5842 5966
Resazurine_A.fum_ATCC46645 196 30 5740 5966
Absorbance_C.alb_MY1055 142 62 3316 3520
Resazurine_C.alb_MY1055 73 35 2338 2446
Resazurine_P.aer_PAO1 + Imipenem 0 0 2480 2480
Resazurine_A.bau_MB5973 0 0 1840 1840
Resazurine_A.bau_MB5973 + Imipenem 25 28 3673 3726
Fluorescence_M.tub_H37Ra 201 44 5721 5966
Grand Total 843 374 41025 42242
1217
PharmaSea Drug Discovery Campaign
1217 Hits (843 Active, 374 Questionable) selected for CherryPiking416 confirmed Hits selected for LCMS Dereplication of knowns
163 samples with no matches in LCMS DB
71 strain-condition-assay selected for confirmation
71 ferment + extraction + assay confirmation
HPLC fractionation+assay+ LCMS and NMR dereplication
20 Strains selected for Big Scale Purification
6016 samples (blanks+extracts+fractions) from 405 strains
52 Compounds
New ikarugamycins
New
New
Biological activity MIC [mg/mL]
Compound ID MRSA C. albicans A. fumigatus
Isoikarugamycin 2-4 2-4 4-8
Ikarugamycin 2-4 4 4-8
28-N-methylIkarugamycin 1-2 4 4-8
30-oxo-28-N-methylIkarugamycin 32-64 >64 >64
Streptomyces zhaozhouensis CA-185989
Mar. Drugs, 2015, 13, 128-140.
New antifungal macrolides
R1= -OH R2= -H R3= -H Compound 1
R1= -OH R2= -OH R3= -H GT-35
R1= -H R2= -OH R3= -SO3H Compound 2
R1= -OH R2= -OH R2= -SO3H Compound 3
MIC values (mg/mL)
Fungal strains Strain number Compound 1 Compound 2 Compound 3 Compound 4
C. albicans ATCC64124 0.5-1 2 0.5-1 8
C. tropicalis ATCC750 0.5-1 2 0.5-1 8
C. glabrata ATCC90030 0.5-1 2 0.5-1 8
C. krusei ATCC6258 0.5-1 1 0.5-1 8
C. parapsilosis ATCC22019 0.5-1 2 0.5-1 16
A. fumigatus ATCC46645 4 4 4 8
Streptomyces caniferus CA-271066
Full absolute configuration of macrolides
Bioinformatic prediction
Bioinformatic + NMR
prediction
Post-Tailoring NMR predicted
Bioinformatic prediction
DISCREPANCY
New spirotetronate derivatives. Phocoenamicins
Micromonospora chaiyaphumensis CA-214671
compounds MIC (mg/mL)ZOI*
mm(mg)
S. Aureus
MB5393
M. tuberculosis
ATCC 25177
M. bovis
ATCC 35734
E. faecium
MB5571
B. subtilis
MB964
phocoenamicin B 8-16 >128 >128 >128 7 (2)
phocoenamicin C 32-64 32 >128 >128 7 (4)
phocoenamicin 4-8 16-32 >128 32-64 7 (4)
vancomycin 2-4 >128
streptomycin 1.6-3.2 0.4-0.8
gentamicin 8 (0.25)
penicillin G 19 (0.06)
Mar. Drugs, 2018, 16, 95.
PRIORITY LIST AND BIOACTIVE COMPOUNDS
Halidrys siliquosa
Antibacterial activity
New bioactive meroterpenoid derivatives
New C-glycosilated angucycline derivatives
Streptomyces sp. CA-237351
Streptocyclinone A Streptocyclinone B
Neuropharmacology, 2018, 141, 283-295
Avda Conocimiento 34, Parque Tecnológico Ciencias de la Salud - 18016 Granada, SPAINTel: +34 958 993 965 Fax: +34 958 846 710
Contact:
Dr. Fernando Reyes
Head of Chemistry